Bioerodable polymeric adhesives for tissue repair

ABSTRACT

Methods for tissue repair are provided employing a matrix comprising a biocompatible, bioerodable polymer, said polymer comprising a thermoplastic lactide-containing terpolymer of monomer units derived from lactic acid, glycolic acid, and either caprolactone or valerolactone, which has a water solubility of about 0.01 to about 500 mg/mL at about 25° C. and adhesive strength of about 600 to about 150,000 Pa and applying the matrix to a tissue defect. The matrix or adhesive can further comprise a filler or a bioactive agent, or both.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. patentapplication Ser. No. 09/956,390, filed Sep. 19, 2001, which is acontinuation of U.S. patent application Ser. No. 08/633,102, filed Apr.16, 1996, now U.S. Pat. No. 6,299,905, issued Oct. 9, 2001, both ofwhich are incorporated herein by reference.

SUMMARY OF THE INVENTION

[0002] In a matrix for tissue repair comprising a biocompatible,bioerodable polymer, this invention provides the improvement wherein thepolymer has a water solubility of about 0.01 to about 500 mg/mL at about25° C. and an adhesive strength of about 600 to about 150,000 Pa so thatthe matrix is tissue adherent. One such matrix comprises a polymer thatalso has a glass transition temperature of less than 0° C. The improvedmatrices are useful for repairing tissues such as bone and cartilage,and for administering biologically active substances.

[0003] These improved matrices may further comprise a filler, abioactive agent, or both.

[0004] In another aspect, this invention provides a pressure sensitiveadhesive for tissue repair comprising (a) a biocompatible, bioerodablepolymer which exhibits adhesive strength of about 600 to about 150,000Pa, (b) a filler and (c) a bioactive agent. Further, this inventionprovides a pressure sensitive adhesive for tissue repair comprising aterpolymer of an α-hydroxycarboxylic acid which exhibits adhesivestrength of about 600 to about 150,000 Pa.

[0005] This invention also relates to methods for repairing bone orcartilage which comprise applying to the bone or cartilage an implantmatrix of this invention.

[0006] In the methods of repairing bone or cartilage using a bioerodableimplant matrix, this invention further provides the improvementcomprising using a tissue-adherent implant matrix of this invention torepair the defect.

[0007] In another aspect this invention relates to implantable articlesof manufacture for use in the release of a bioactive agent into aphysiological environment, said articles comprising a biologicallyactive agent disbursed in an implant matrix of this invention.

DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention is directed to improvements in matrices fortissue repair comprising biocompatible, bioerodable polymers. In oneimprovement, the matrix comprises a polymer which has a water solubilityof about 0.01 to about 500 mg/mL at about 25° C. and an adhesivestrength of about 600 to about 150,000 Pa so that the matrix is tissueadherent. One such matrix comprises a polymer that has a glasstransition temperature of less than 0° C. The improved matrix canfurther comprise a filler or a bioactive agent, or both. An especiallyuseful attribute of the improved matrices is that the matrix adheres totissues such as bone or cartilage. In addition, the matrix has a texturelike that of dough or putty; thus, it is particularly suitable for beingmolded to fit into a site needing repair.

[0009] In another aspect, this invention provides pressure sensitiveadhesives for tissue repair comprising (a) a biocompatible, bioerodablepolymer which exhibits adhesive strength of about 600 to about 150,000Pa, (b) a filler and (c) a bioactive agent. Further, this inventionprovides pressure sensitive adhesives for tissue repair comprising aterpolymer of an α-hydroxycarboxylic acid which exhibits adhesivestrength of about 600 to about 150,000 Pa.

[0010] The implant matrices and adhesives of this invention can beapplied to the bone-contacting surfaces of prosthetic appliances (as acement), or they can be inserted into and around bone defects andcavities or cartilage surfaces (as a filler). The matrix or adhesivebiodegrades gradually. As it biodegrades, it is replaced by developingbone or cartilage tissue in a manner which permits a natural healing ofthe tissue. Thus, it provides an effective means for treating orrepairing bone or cartilage.

[0011] When the matrix or adhesive further comprises a bioactive agent,it serves as a depot device for release of the bioactive agent. Releaseof the agent occurs as the matrix or adhesive biodegrades afterimplantation.

[0012] Many attempts have been made to develop a repair matrix thatcould facilitate bone or cartilage repair and also deliver bioactiveagents such as growth factors. Such a matrix could be used instead ofbone grafts. Thus far, only matrices comprised of natural products suchas collagen have shown promise. Collagen, however, is difficult tomanufacture and control in order to meet regulatory standards. Inaddition, surgeons are not satisfied with collagen matrices because theyare difficult to form and/or handle.

[0013] Other approaches to replace bone grafts have includedconventional bioresorbable polymers, ceramics such as tricalciumphosphate (TCP), natural polymers, such as collagen, proteoglycans,starches, hyaluronic acid and modified bone matrix. To date theseefforts have only produced delivery matrices which (a) impede healing,(b) provoke negative tissue reactions, (c) cannot be sterilized, (d) aredifficult to use or (e) cannot be manufactured to the satisfaction ofregulatory bodies.

[0014] For example, one approach was to use conventional bioresorbablepolymers such as polylactide-co-glycolide (PLG) to administer growthfactors. It was very difficult, however, to combine PLG with the growthfactor without inactivating the growth factor. Other disadvantagesencountered with PLG were that, when it was implanted, it inhibited thebone healing response and occasionally caused aseptic sinus tract andinflammation and destroyed surrounding bone.

[0015] Another attempt to develop an effective bone repair matrixinvolved implanting a bone growth factor absorbed on a ceramic such asTCP. The problem with this approach was that the TCP particles migratedout of the defect area too quickly to deliver the growth factoreffectively.

[0016] A major problem encountered with previously tried deliverysystems is that the bioerodable material could not be mixed with thegrowth factor prior to the time of surgery. Mixing the delivery matrixwith the bioactive material immediately prior to, or during, the surgeryprocess is very awkward and can lead to inconsistent results.

[0017] The bioerodable matrices and adhesives of this invention solveseveral of the problems encountered with previous delivery systems. Theyare especially useful in the delivery of bioactive proteins such asgrowth factors because the polymer component dissolves in solvents whichare compatible with proteins. Thus, it is possible to formulate thebioactive component in the polymer adhesive matrix in advance, i.e.,well before a surgical procedure, under acceptable regulatoryconditions, including sterilization of the product without inactivatingthe bioactive components. Quality control during the preparation ofdelivery systems using the present adhesive products is, therefore,greatly improved.

[0018] Other advantages of the polymer implant matrices and adhesives ofthis invention are that they are biocompatible and bioerodable in vivo.The term “biocompatible” means that the polymer is non-toxic,non-mutagenic and, at most, elicits only a minimal to moderateinflammatory reaction. The term “bioerodable” means that the polymereither degrades or is resorbed after implantation into products that areused by, or are otherwise eliminated from, the body by existingbiochemical pathways.

[0019] The present matrices comprise polymers that are bioerodablewithin a period of from about three hours to about two years. Thisperiod can be varied, depending upon the desired application. Apreferred period is from about one day to about one month; anotherpreferred period is from about two weeks to about three months. Theperiod for bioerosion is the time after which the polymer will no longerbe detectable at the site of implantation, using standard histologicaltechniques.

[0020] Thus, an important advantage of the present polymer implantmatrices is that a second surgical procedure to remove the matrix is notrequired because it degrades with time, and its degradation products areabsorbed by the body.

[0021] One required feature of certain of the adhesive bioerodablepolymers useful in the improved matrices of this invention is theirwater solubility. They are soluble in water at about 0.01 to about 500mg/mL of water at about 25° C. (ambient temperature). Typically, thepolymers are soluble in water at about 0.1 to about 500 mg/mL of water.Preferably they are soluble at about 5 to about 400 mg/mL of water.

[0022] Some investigators have reported aseptic necrosis, inflammation,or sinus tracts in animals where poly(α-hydroxycarboxylic acid) implantshave been used. It is generally thought that these adverse reactionswere caused by local acidosis from the degradation of the polymer. Useof more soluble ionomer forms of the polymers avoids the danger ofdeveloping local acidosis at implant sites because the polymers dissolveand are diluted or carried away before quantities of acidic degradationproducts are produced.

[0023] This water solubility allows the polymers to be more readilydissolved by serum at the surface of the implant matrix and thereafterdistributed into surrounding body fluids where they can be mobilized forhydrolysis at remote sites. This feature is important because hydrolysisof some polymers results in a localized pH gradient which can be adverseto local cell growth. Hydrolysis occurring at the implant site producesan unnatural concentration of hydrolysis products (and increasedacidity) at the surface of the matrix. Such acidity can easily interferewith ongoing tissue repair. The water soluble polymers used in theimproved matrices of this invention, therefore, preserve conditions thatoptimize a localized environment for cell viability and growth at theimplant surface.

[0024] Certain polymers used in the matrices of this invention, thepolyesters, have a glass transition temperature (Tg) of less than 0° C.When used with a filler, polymers with a Tg of less than 0° C. haveexcellent handling properties.

[0025] A required feature of all the polymers for use in the matricesand adhesives of this invention is a threshold level of adhesiveness.Adhesiveness has been found to be important for optimizing implantperformance. Adhesiveness is an intrinsic property that is not readilycorrelated with polymer properties, but can easily be assessedempirically. Adhesiveness is a characteristic that derives from a widevariety of polymer parameters, including polymer type, i.e., the natureof the covalent linkages linking the monomers, molecular weight andintrinsic structure and as well the nature of the surface to which thematrix will be adhered. Skilled practitioners in the art can readilyassess polymer adhesive properties using known techniques, such as thoseillustrated in the examples infra.

[0026] The polymers used in the matrices and adhesives of this inventionexhibit adhesive properties on different substrates, such as, forexample, dry substrates like glass and water-swollen poly(2-hydroxyethylmethacrylate) (“pHEMA”) on glass, which simulates wet tissues.Typically, the polymers withstand a maximum stress on a glass substrateof about 1,000 to about 150,000 Pa, preferably about 10,000 to about40,000 Pa, and most preferably, about 12,000 to about 16,000 Pa. Thepolymers withstand a maximum stress on a pHEMA substrates of about 600to about 90,000 Pa, preferably about 2,500 to about 40,000 Pa, and mostpreferably about 5,500 to about 8,500 Pa. Thus, the range of adhesivestrength is from about 600 to about 150,000 Pa.

[0027] The polymers are moldable by hand at a temperature of about 60°C. or below. Typically, they are moldable at about 4° C. to about 60°C., preferably at about 15° C. to about 50° C., and most preferably atabout 20° C. to about 30° C. The degree of moldability at a selectedtemperature is dependent upon the characteristics of the polymerselected as well its molecular weight. The matrix containing the polymerremains moldable after it has been implanted within the body.

[0028] A variety of polymers can be used in the matrices and adhesivesof this invention. The polymers must be biocompatible and susceptible torapid biodegradation in order to be replaced by new tissue. The polymersmay be homopolymers, terpolymers, copolymers, blocked copolymers, orblends of polymers. Bioerodable polymers include polyanhydrides,polyorthoesters, polyesters (such aS polylactic acid (PL), polyglycolicacid (PG), polyhydroxybutyric acid, polymalic acid, polyglutamic acidand polylactones) and poly(amino) acids.

[0029] One type of polymer especially useful in the matrices andadhesives of this invention is a polyester ionomer, more particularly, anon-toxic salt of a bioerodable carboxy-terminated polyester of thegeneral formula RO˜PE˜COOH or HOOC˜PE˜COOH wherein R is hydrogen orC₁-C₄ alkyl and ˜PE˜ is a divalent residue of a polyester. The polyestercan comprise a homopolymer, copolymer, or terpolymer of biocompatiblehydroxy acids, for example, lactic acid, glycolic acid, ε-hydroxycaproicacid, and Γ-hydroxyvaleric acid. Alternatively, the polyester can beformed using copolymerization of a polyhydric alcohol and abiocompatible polycarboxylic acid. Most typically such copolymers areformed between dihydric alcohols, for example, propylene glycol forbiocompatibility and biocompatible dicarboxylic acids. Representativecarboxylic acids for formation of the polyesters useful for preparingthese polyester ionomers include a Kreb's cycle intermediate such ascitric, isocitric, cis-akonitic, α-ketoglutaric, succinic, maleic,oxaloacetic and fumaric acid. Many of such carboxylic acids haveadditional functionalities which can enable further cross-linking of thepolymers if desirable.

[0030] The polyesters can be further modified, for example, by reactionwith a cyclic carboxylic anhydride to convert residual hydroxyfunctionality to the carboxy-terminated forms useful for preparation ofthese polyester ionomers.

[0031] The carboxy-terminated polyesters used to prepare the polyesterionomers are selected to have a threshold water solubility between about0.01 and about 500 mg/mL of water, preferably about 0.5 to about 350mg/mL of water, at ambient temperature. The polyester precursors have aweight average molecular weight of about 400 to about 10,000, moretypically about 1,000 to about 5,000. Conversion of these compounds byneutralization with pharmaceutically acceptable bases produces polyesterionomers having enhanced water solubility relative to thecarboxy-terminated polyester precursors but retaining other polymerfunctionality.

[0032] The polyester ionomers are prepared from mono- orbis-carboxy-terminated polyesters. Generally, the carboxy-terminatedpolyester is dissolved in an organic solvent and neutralized by theaddition of a stoichiometric amount of a physiologically acceptablebase. In one embodiment, the neutralization is carried out with lessthan a stoichiometric amount of base to produce a composition comprisinga carboxy-terminated polyester and its corresponding ionomer, the ratioof those components being dependent on the degree of neutralization.Suitable bases for use in forming the polyester ionomers includehydroxides of Group Ia or Group IIa metals including preferably thehydroxides of lithium, sodium, potassium, magnesium and calcium, as wellas physiologically compatible salt-forming amines. Followingneutralization of the carboxy-terminated polyester, the resultingionomer can be isolated using standard isolation techniques. the ionomeris typically dried prior to use in fabrication of implant matrices andadhesives.

[0033] The carboxy-terminated polyesters can be prepared usingart-recognized procedures for polyester synthesis. The carboxy-terminus(or termini) on such compounds can be formed by reaction of hydroxyfunctional polyesters with, for example, a stoichiometric amount of acyclic anhydride of a C₁-C₆ dicarboxylic acid, such as succinicanhydride.

[0034] Bis-hydroxy functional polyesters are readily prepared byreaction of a dihydric alcohol initiator, for example, propylene glycolor ethylene glycol, with one or more cyclic hydroxy acid esters, forexample lactide, glycolide or caprolactone. Reaction of such bis-hydroxyfunctional polyesters with cyclic anhydrides produces bis-carboxyfunctional polyesters that can be used to prepare the ionomers describedsupra.

[0035] The polyester prepolymers used for the preparation of theionomers can be prepared using art-recognized polyester-forming reactionchemistry, typically using, for example, metal catalysts to promoteester-forming reactions. One problem with the prior art procedures isthe difficulty in removing the metal catalyst from the productpolyesters. Removal of the catalyst is particularly crucial when thepolyesters are intended for use in medical applications.

[0036] It has been found that polyesters of hydroxy acids can beprepared in high yields and high purity with good control overstructure/functionality by reacting the corresponding cyclic esters witha hydroxy functional initiator at elevated temperatures undersubstantially anhydrous conditions. Thus, one preferred method forpreparing the polyesters consists of reacting an initiator, for example,a mono-hydric or dihydric alcohol, with at least one cyclic hydroxy acidester under substantially anhydrous conditions at elevated temperatures.The reaction is preferably carried out neat (an absence of solvent) at atemperature of about 100-180° C., more preferably about 120-160° C. Theterm “substantially anhydrous conditions” means that routine efforts aremade to exclude water from the reaction mixture and can typicallyinclude such steps as pre-drying the reaction vessel with heat andcarrying out the reaction under drying conditions.

[0037] The structure of the polyester is controlled by selection andstoichiometry of the cyclic hydroxy acid ester reactant(s) and theamount of initiator used with lower relative initiator amounts leadingto higher average molecular weight product and higher relative amountsof initiator leading to lower average molecular weight product.

[0038] The hydroxy functional initiator can either be a monohydricalcohol, for example a C₁-C₄ alkanol, or a di-or polyhydric alcohol.Alternatively, the hydroxy functional initiator can be a hydroxy acid,for example glycolic acid. The product hydroxy-terminated polyesters canbe converted to a carboxy-terminated polyester that can be used toprepare the polyester ionomers by reaction with a stoichiometric amountof a cyclic anhydride.

[0039] The method for preparing polyester polymers for use in preparingthe polyester ionomers can be carried out as well in the presence of acyclic carboxylic acid anhydride to provide directly the correspondingcarboxy terminated polyester compound. The reaction is carried out underthe same conditions described supra for preparing the polyester. Mosttypically the reaction is carried out using about equimolar amounts of amonohydricalcohol initiator and the cyclic anhydride. Where theinitiator is a dihydric alcohol, the molar ratio of the cyclic anhydrideto the initiator is preferably raised to about 2:1.

[0040] Preferred polyester ionomers are those made up of lactide,glycolide and caprolactone or valerolactone. Polymers oflactide/glycolide/caprolactone (PLGC) are especially beneficial. PLGCterpolymers having a molecular weight in the range of about 1,000 to3,000 are especially preferred. Terpolymers wherein the lactide andglycolide each make up about 35-45% of the terpolymer, and thecaprolactone or valerolactone make up about 10 to about 30% of theterpolymer are particularly useful.

[0041] Selected poly(amino acids)are another type of polymer useful inthe matrices and adhesives of this invention. Certain poly(amino acids)exhibit adhesive properties toward connective tissue, such as cartilageand bone. The poly(amino acid) can be: (1) a classic poly(amino acid) ofthe formula H₂N—Q—COOR₂ in which Q is the divalent residue of apolypeptide and R₂ is H, a metal cation, or ammonium, or (2) apseudo-poly(amino acid).

[0042] The matrix may comprise two or more different poly(amino acids),each of the formula H₂N—Q—COOR₂ wherein:

[0043] Q is a divalent residue of a polypeptide formed from 1 to 3species of amino acids;

[0044] the amino acid components of Q are represented by the formulaαX+bY+cZ;

[0045] wherein a, b, and c represent the respective mole fractions ofthe amino acids X, Y, and Z; a=0 to 1, b=0 to 1, and c>0 but <1; anda+b+c=1.0;

[0046] X is selected from glutamate, asparagine, aspartate, andglutamine;

[0047] Y is selected from lysine and arginine; and

[0048] Z is selected from cysteine, methionine, serine, threonine,glycine, alanine, valine, leucine or isoleucine.

[0049] Alternatively, the matrix may comprise a divalent or multivalentmonomer and a poly(amino acid) of the formula H₂N—Q—COOR₂ as definedsupra wherein the Q polypeptide is formed from 1 to 3 species of aminoacids.

[0050] A wide variety of polypeptides in a wide variety of ratios may beused to form the useful poly(amino acids). The polypeptides areavailable commercially from Sigma Chemical Company, P.O. Box 14508, St.Louis, Mo. 63178.

[0051] Certain amino acid homopolymers, however, are not useful in thematrices. For example, amino acids with aliphatic side chains do notinteract well enough with biological surfaces. They may, however, beused as chain extenders or modulators, along with cysteine, methionine,serine, and threonine, in mixed polymers. Amino acids with aromatic sidechains exhibit low rates of diffusion in the body and are, therefore,not suitable to be components of selected poly(amino acids). Histidineis also not a suitable component due to its limited interaction withbiological surfaces. Histidine may, however, be used to complex with thepolyamino acids as a monomer.

[0052] Particular divalent or multivalent monomers may be used incombination poly(amino acids) in the matrices. Amino acids with two ormore positive charges at physiological pH, such as lysine, arginine, orhistidine, form complexes with poly(amino acids) bearing negativecharges at physiological pH. Likewise, amino acids with two or morenegative charges, such as aspartate or glutamate, can form complexeswith poly(amino acids) bearing positive charges.

[0053] In the pseudo-poly(amino acids) that can be selected, thedipeptide monomers are covalently bound through other than normalpeptide linkages. Pseudo-poly(amino acids) suitable for use are thosehaving the requisite adhesive character. They can be prepared using thechemistry described, for example, in Kohn, J. and Langer, R.,Polymerization Reactions Involving the Side Chains of α-L-Amino Acids,J. Amer. Chem. Soc., 109, 917 (1987) and Pulapura, S. and Kohn, J.,Biomaterials Based on “Pseudo”-Poly(Amino Acids): A Study ofTyrosine-Derived Polyiminocarbonates, J. Polymer Preprints, 31, 23(1990), which are incorporated by reference. The pseudo-poly(aminoacids) can be used alone or in combination with a classic poly(aminoacid) or with a different pseudo-poly(amino acid).

[0054] As discussed supra, the composition of the polymer, as well asthe molecular weight and physical properties, can be varied. Those inthe art will also appreciate that compounds can be mixed into, orpolymerized with, the polymer as required for additional strength orother desirable physical properties, using materials known in the art.For example, TCP or other ceramic-type materials that provide increasedviscosity can be added to the composition.

[0055] The dissolution rate of polymers such as the PLGC terpolymers canbe varied by end group modification. For example, PLGC terpolymers withOH end groups degrade very slowly; PLGC terpolymers wherein the OH endgroups have been partially neutralized, e.g., by neutralization of about40 to 60% of the end groups with sodium hydroxide, degrade at amoderately slow rate; and PLGC terpolymers wherein most the OH endgroups have been neutralized, e.g. by sodium hydroxide, degrade within afew days. Exemplary end groups are OH and COONa+, but any ion orfunctional group that can be placed on the polymers could be used. Theamount of end group modification can have a dramatic effect on thedissolution rate.

[0056] In addition to end group changes, variations of molecular weightand composition can be selected to prepare suitable compositions.Increases in molecular weight increase the time to dissolution. Also,blending in a high MW polymer will increase the time to dissolution, orblending in a low MW polymer will decrease the time.

[0057] In general, when the matrix is used to repair bone defects, thepolymer is selected to degrade over a period of three hours to twoyears. Preferably, the polymer will degrade in about one month, mostpreferably in about two weeks. The desired degradation time will dependon the nature of the repair site, including the local tissue type, thesupport function being served by the implanted matrix, and the natureand concentration of the bioactive component, if any, in the implantmatrix. Targeted degradation times can be achieved by selection ofpolymer/filler combinations on an individual basis.

[0058] In the matrix, the polymer may be combined with a bioactiveagent, one or more fillers, or both. When the matrix contains a filler,it typically contains about 1 to about 90 weight percent filler,preferably about 30 to about 70 weight percent, and most preferablyabout 35 to about 50 weight percent of filler.

[0059] The filler may be particulate, fibrous, organic, inorganic or amixture of organic and inorganic. Suitable fillers include bone chips,tricalcium phosphate, hydroxylapatite (“HA”), small intestine submucosa(“SIS” as described in U.S. Pat. Nos. 4,902,508, issued Feb. 20, 1990,and 4,956,178, issued Sep. 11, 1990), bioglass granules, syntheticpolymers, calcium carbonate, calcium sulfate and collagen, or otherextracellular matrix compound, or various mixtures thereof.

[0060] When the filler is particulate, the average particle size is fromabout 20 μm to about 2,000 μm, more preferably about 75 to about 700 μm,and most preferably, about 100 μm to about 500 μm.

[0061] As discussed supra, the implant matrix may contain a bioactiveagent or agents. A bioactive agent is a compound or material thataffects the living cells in its surrounding environment, e.g., it actsto enhance the healing process.

[0062] Bioactive agents preferred for use in the present invention aregrowth factors, growth factor binding proteins or cells. Examples ofsuitable growth factors include: a fibroblast growth factor, atransforming growth factor (e.g., TGF-β₁), a bone morphogenetic protein,epidermal growth factor, an insulin-like growth factor or aplatelet-derived growth factor.

[0063] Examples of growth factor binding proteins are insulin-likegrowth factor binding proteins (IGFBP's) such as IGFBP's 3 and 5.Examples of suitable cells include bone marrow cells and mesenchymalstem cells. The bioactive material can also be an osteogenic agent whichstimulates or accelerates generation of bone upon implantation into abone defect site. Examples of osteogenic agents include demineralizedbone powder, morselized cancellous bone, aspirated bone marrow, boneforming cells, and other bone sources.

[0064] The bioactive agent may also be an antibacterial substance.Examples of useful antibacterial agents include gentamicin andvancomycin.

[0065] When a bioactive agent is included in the matrix or adhesive, itis incorporated in amounts of from about 10⁻⁵% to about 33% by weight ofthe matrix. Typically, the agent is incorporated at a rate of from about10⁻²% to about 20% by weight of the matrix. A preferred rate ofincorporation is from about 10⁻¹% to about 5% by weight.

[0066] When the bioactive agent is a growth factor, it is generallyincorporated into the matrix or adhesive in amounts from about 10⁻⁵% toabout 1% by weight of the matrix. When cells are the active component,the range is from about 0.5% to about 50% by weight. When using an agentsuch as demineralized bone, bone marrow and the like, the range ispreferably from about 5% to about 95% by weight. For TGF-β₁ thepreferred range is from about 10⁻⁴% to about 0.05% of TGF-β₁ by weightof the matrix.

[0067] The percent of bioactive agent should be such that it willrelease from the implanted matrix in vivo in an effective manner,generally over a period of from about a day to about 30 days and longer,depending on the nature and application of the composition.

[0068] The release rate of a bioactive agent, such as TGF-β₁, can bevaried by modification of the polymer as discussed supra, e.g., byvarying its end groups, molecular weight or composition.

[0069] Other agents that may be added to the matrix include: an extractfrom whole blood, packed red cells, platelets, plasma (fresh or freshfrozen), serum, skin, bone, cartilage, tendon or microorganisms;synthetic proteins, etc. Suitable proteins can be any one of a widevariety of classes of proteins, such as keratins, collagens, albumins,globulins, hormones, enzymes, or the like. The material can be simplepeptides, simple proteins, or conjugated proteins, such asglycoproteins, mucoproteins, lipoproteins, heme proteins,nucleoproteins, or the like.

[0070] Antioxidants may also be included in the matrix. Antioxidantssuitable for use include tocopherol, citric acid, butylatedhydroxyanisole, butylated hydroxytoluene, tert-butylhydroquinone, propylgallate, sodium ascorbate, and other antioxidants which are “generallyrecognized as safe” by the Food and Drug Administration.

[0071] Thus, the implant matrices can be prepared by blending thepolymer with one or more bioactive agents and optionally otherexcipients, for example, additives to optimize retention of biologicalactivity and polymer functionality during sterilization, and then bysterilizing and packaging the implant formulation for surgical use.

[0072] Sterilization can be accomplished by radiation with about 1 toabout 3 mRad of gamma radiation or electron beam radiation. If thebioactive agent is a protein or peptide, biological activity can beoptimized during sterilization by including in the formulation 1) anextraneous protein, for example albumin or gelatin; and 2) a freeradical scavenger (antioxidant), for example propyl gallate,3-tert-butyl-4-hydroxyanisole (BHA) or ascorbic acid, in amountseffective to retard radiation-induced degradation of the biologicallyactive peptide. The sterilization is preferably conducted at lowtemperature, for example −70° C.

[0073] When a filler is used in the matrix with a biologically activepeptide or protein, it is advantageous to form a mixture of thebiologically active compound and an extraneous protein such as albuminor gelatin, and coat the filler with that formulation prior to blendingthe filler into the polymer.

[0074] Preferred matrices for bone repair include the following: Rangeof Most Preferred Preferred Ingredient Amount (mg) Amount (mg) TCP (orSIS) 100   10-500 Polymer* 200   20-500 Gelatin 10  1-100 TGF-β₁ and/or 10⁻²  10⁻⁴-10⁻¹ Cells 100   10-200 Antioxidant  2  0.5-50

[0075] The implant matrices of this invention can be prepared usingstandard formulation techniques. If the matrix includes a bioactiveagent, the polymer can be mixed with the agent or used to encapsulateit, again using known methods such as mixing and compressing andmicroencapsulation.

[0076] This invention also provides an implantable article ofmanufacture for use in the release of a bioactive agent into aphysiological environment comprising a biocompatible tissue-adherentimplant matrix of this invention and one or more bioactive agents.Preferred implantable articles are those wherein the bioactive agent isa growth promoting factor.

[0077] Although the polymers have been described for use in repairingtissues such as bone and cartilage and in a delivery matrix for abioactive agent in vivo, these descriptions are illustrative only andare not intended to be limiting in any way. There are many otherapplications for the bioerodable adhesive polymers of this invention.

[0078] For example, the polymers can be used in the treatment of bonetumors. Such treatment typically involves excision of the tumor as wellas portions of the surrounding bone, leaving a large cavity in the bone.A graft using autogenous bone (bone harvested from another site in thepatient's body) is the conventional and accepted technique for fillingsuch bony defects. Although use of autogenous bone provides rapidincorporation of new bony ingrowth into a bone cavity, this procedure isassociated with a morbidity caused by the required surgical exposureneeded to harvest the patient's bone. Moreover, some patients,particularly osteoporotic individuals, have very limited amounts of bonethat are appropriate for use as a graft.

[0079] Alternatively, allografts, i.e., bones taken from otherindividuals, may be used as bone-grafting material. There are certainrisks associated with such allografts, however, including the transferof infections and even unrecognized malignant cells from the harvestedpatient to the grafted patient as well as the problem of immunologicbarriers between all individuals. Furthermore, these processes arecomplicated and labor-intensive. Thus, the implant matrices of thisinvention offer a distinct improvement over traditional treatments forbone tumors.

[0080] The polymers used in the matrices and adhesives of this inventionare typically prepared so that they form a viscous adhesive rather thana conventional solid. When the polymer is mixed with a particulatefiller to form the biocompatible matrix or adhesive, the polymer can beused to coat the particles of filler. An example of a suitableparticulate filler is a ceramic such as TCP. When the particles arecoated with the polymer adhesive, they form a self-adherent dough-likesubstance that can be conveniently molded to fit surgically into bonedefects. When a bioactive agent, such as a protein growth factor, is tobe included in the matrix, it can be absorbed onto the particles of thebiocompatible solid filler prior to being coated with the polymeradhesive.

[0081] This invention also relates to improvements in methods ofrepairing bone or cartilage using a bioerodable implant matrix, whereinthe improvement comprises using a tissue-adherent matrix of thisinvention to repair the bone or cartilage.

[0082] Preferred improvements are those wherein the matrix contains abioactive agent, particularly those wherein the bioactive agent is agrowth-promoting factor.

[0083] When using a matrix of this invention to repair bone orcartilage, a surgeon, physician or other caregiver first determines thesize of the cavity or void to be filled, or the dimensions of the repairsite, and removes the appropriate amount of polymer adhesive matrix frompackaging. Typically, the packaging is a barrier package which preventswater vapor from contacting the polymer in the composition; however, itis understood that the packaging may be any one of a wide variety ofcontainers.

[0084] Following removal from the packaging, the surgeon then molds theadhesive implant matrix at ambient temperature into dimensionscompatible with the repair site. In the case of bone repair, the matrixis molded to the dimensions of the cavity or void to be filled. In thecase of connective tissue repair, it is molded to fit the dimensions ofthe repair site. The adhesive matrix is then applied to the cavity orrepair site in a manner which permits it to adhere to the bone orcartilage for a time sufficient to effect its repair. Typically, thesurgeon presses the molded matrix against the damaged, and often wet,tissue. Because the matrix has adhesive properties, when it is appliedto the surrounding bone or connective tissue with pressure, it willstick and remain in place long enough to effect repair of the bone ortissue.

[0085] When the matrix contains a bioactive agent, it is typicallyimplanted in a site in the body where a concentration of the bioactiveagent would be beneficial. Thus, for example, in the treatment of anosteoporosis-induced fracture involving a void or bony defect, animplant matrix containing a growth-promoting agent is molded to conformto the bone defect or cavity and is inserted by the surgeon at thatlocation. Similarly, the matrix can be implanted or injected into softtissue for sustained drug release.

[0086] Preparation

[0087] Poly(lactide/glycolide/ε-caprolactone) Ionomer

[0088] Instrumentation. Gel permeation chromatography (GPC) was used todetermine molecular weights and molecular weight distributions, Mw/Mn,of polymer samples with respect to polystyrene standards (PolysciencesCorporation). The system configuration was described by R. F. Storey andT. P. Hickey, J. Polymer Sci. 31, 1825 (1993). Throughout thisspecification, unless otherwise stated, the term “molecular weight”refers to weight average molecular weight.

[0089] General Procedure

[0090] 1. Synthesis of Acid-Terminated Polymers:

[0091] Glassware was dried at 145-155° C. for 24 h, fitted with rubbersepta, and cooled under a flow of dry nitrogen. Polymerizations were runin 250-mL Erlenmeyer flasks with 24/40 ground glass joints sealed withevacuated glass stoppers wrapped with teflon tape. To a flask (250 mL)containing a magnetic stir bar were added D,L-lactide (18.17 g,1.26×10⁻¹ mol), glycolide (14.63 g, 1.26×10⁻¹ mol), ε-caprolactone (7.20g, 6.30×10⁻² mol), glycolic acid (1.66 g, 2.18×10⁻² mol), succinicanhydride (2.19 g, 2.18×10⁻² mol). The flask was purged with nitrogenand heated in a 135° C. constant temperature bath for 20 h withcontinuous stirring. At 65 h of reaction, the temperature was lowered to110° C. The polymerization was allowed to proceed for 146 h and was thenquenched in an ice-water bath.

[0092] 2. Analytical Titration Procedure (2,000 g/mol Sample):

[0093] To a 125-mL Erlenmeyer flask was added a (˜2,000 g/mol) polymersample (0.30-0.40 g). The polymer sample was completely dissolved in THF(50 mL), and water (15 mL) was added to the solution. Phenolphthalein (1g/100 mL MeOH) (5 drops) was added to the polymer solution, and theflask was placed in an ice bath. The sample was titrated with an aqueoussolution of NaOH (0.5047 N) to a light pink end point. An averageequivalent weight was calculated from the values of at least threetitrations.

[0094] 3. Bulk Polymer Titration Procedure (2,000 g/mol Sample):

[0095] To a 1,000-mL Erlenmeyer flask was added a (˜2,000 g/mol) polymersample (34.32 g), and the polymer was dissolved in THF (450 mL). Theaverage equivalent weight from analytical titration procedure 2, supra,was used to calculate the exact amount of titrant (85.3 mL, 0.5047 Naqueous NaOH) necessary to completely neutralize the polymer sample.This amount was slowly added to the polymer solution as it was stirredin an ice bath.

EXAMPLES Example 1 Determination of Adhesive Properties

[0096] General Procedure: The adhesion characteristics of the polymerswere determined in a tensile test in accordance with the followingprocedure:

[0097] Glass microscope slides were cleaned by first immersing them in ahot sulfuric acid bath for 10 minutes. The slides were rinsed thoroughlywith ultrapure water. Then they were placed in a warm ammoniumhydroxide:hydrogen peroxide (4:1 by volume) bath for 1 minute. Theslides were again rinsed with ultrapure water and dried with filterednitrogen. The clean glass slides are the dry glass substrates.

[0098] Each slide was placed on a holder that exposed 4.84 cm² of area.An aqueous solution of 3% by weight of polymer in nano-pure water wasplaced on this exposed area, and the slide was dried under vacuum. Allsamples were stored in a desiccator before mechanical testing.

[0099] Comparison slides for testing adhesion to wet surfaces were madeusing poly(2-hydroxyethyl methacrylate) (pHEMA). The films of pHEMA wereformed using a 4% by weight solution of polymer in methanol, using theprocedure described supra. The solution was dried with nitrogen gasfollowed by 3 hours vacuum.

[0100] Mechanical tests were made using a Series 4400 Instron. In orderto test the adhesive properties of the polymer film, the glass slidewith the test polymer was pressed on a clean dry glass slide with aforce of 5 Newtons for 5 minutes. The Instron was then used to measurethe stress and strain at which the two glass slides separated by beingpulled apart at an angle of about 90° relative to the face of the slide.The separation speed was 0.5 mm per minute.

[0101] A separate adhesion test was carried out on the slides made withswollen pHEMA in order to simulate a wet tissue surface. The pHEMA film,cast on glass, was placed in a 100% humidity chamber for 30 minutesbefore testing. The glass slide with the test polymer was then pressedon the pHEMA slide for 5 minutes at 5 Newtons.

[0102] A. Test Results for Homopolymers:

[0103] The results of these tests with illustrative poly(amino acid)homopolymers are summarized in Table 1. TABLE 1 HOMOPOLYMERS GlassSwollen pHEMA max. max. max. max. Polymer* (MW) stress (Pa) strainstress (Pa) strain pGlu (1,000) 6500 0.60   0 0 pGlu (15,300) 3400 0.651000 0.20 pLys (22,700) 2800 0.30  650 0.12 pLys (42,000) 10000  0.702300 0.23 pGln (3,500) 9000 0.85   0 0

[0104] Surprisingly, various homopolymers, such as pGlu (15300), pLys(22700), and pLys (42000) were found to stick to the pHEMA. It was foundthat all of the homopolymers adhere to the glass surface.

[0105] It was determined that the adhesive strength of differentmaterials may be manipulated by changing the homopolymer and/or themolecular weight of the homopolymer. These results can be extrapolatedto other amino acids of their class which would be useful in thesecompositions. In addition, the homopolymers could be substituted withmixed polymers such as copolymers, a terpolymer, block copolymers, ormixtures thereof.

[0106] B. Test Results of Polymer-Monomer Complexes:

[0107] The results of these tests with typical polymer-monomer complexesare illustrated in Table 2. TABLE 2 POLYMER-MONOMER COMPLEXES GlassSwollen pHEMA max. max. stress max. stress max. Complex (MW)[Wt. Ratio](Pa) strain (Pa) strain pGlu(1000):Lys[2:1] 1100 0.12 1000 0.07pGlu(1000):Lys[1:2] 9000 0.60 0 0 pGlu(15300):Lys[2:1] 8000 0.60 23000.40 pGlu(15300):Lys[1:1] 10000 0.80 1500 0.16 pLys(22700):Glue[1:1] 0 00 0 pLys(22700):Glue[1:2] 0 0 0 0 pLys(42000):Glue[1:0.8] — — 5500 0.50pLys(42000):Glue[1:2] 0 0 1150 0.25 pGln:Lys(35000):Lys[1:0.6] 3500 0.301250 0.13 pGln:Lys[1:0.7] 4800 0.65 0 0 pGln:Lys[1:1] 0 0 1200 0.20pGln:Glue[1:1] 5000 0.45 0 0 pGln:Glue[1:2] 3000 0.30 0 0

[0108] It was found that the adhesion of pGlu polymers (1000 and 15300)on glass improved as the amount of Lys monomer was added. On swollenpHEMA, a higher pGlu to Lys monomer ratio favored adhesion. The adhesionof pLys (22700 and 42000) on glass decreased as the amount of Gluemonomer increased. Finally, the addition of Glue monomer improved theadhesion of pLys (42000) to swollen pHEMA.

[0109] These results demonstrate how a specific adhesive strength todifferent types of material may be achieved. The type of amino acidhomopolymer used, or mole weight of the homopolymer, can be tailored toproduce a desired adhesive property to different materials. Thehomopolymers could be substituted with mixed polymers such ascopolymers, a terpolymer, block copolymers, or mixtures thereof.

[0110] C. Test Results for Polymer Blend Complexes:

[0111] The results of these tests with typical polymer blend complexesare illustrated in Table 3. TABLE 3 POLYMER BLENDS Glass Swollen pHEMABlend of pGln (3500) with max. max. amino acid homopolymer (MW) stressmax. stress max. [Wt. Ratio] (Pa) strain (Pa) strain pGlu(1000) [1:0.4]3000 0.40 0 0 pGlu(1000)[1:0.8] 5000 0.55 3300 0.25 pGlu(1000)[1:2] 28000.55 0 0 pGlu(15300)[1:0.9] 7500 0.60 2300 0.30 pGlu(15300)[1:2] 160001.50 8500 1.00 pLys(22700)[1:0.3] 2000 0.20 0 0 pLys(22700)[1:0.8] 130000.95 0 0 pLys(22700)[1:2] 6000 0.42 0 0 pLys(24000)[1:0.5] 1800 0.25 0 0pLys(42000)[1:0.8] 9000 0.85 5500 0.90 pLys(42000)[1:0.84] 11000 1.75 —— pLys(42000)[1:0.9] 8000 1.40 — — pLys(42000)[1:1.2] 10000 0.90 — —pLys(42000)[1:1.25] 13000 2.30 9000 1.10 pLys(42000)[1:2] 3100 0.18 35000.55

[0112] It was discovered that polymer blends of pGln with pGlu (15300)showed a noticeable improvement over pGln homopolymer (see Table 1) inadhesion and strength and strain on both substrates. The samplepGln:pGlu (15300) [1:2] was one of the most preferred adhesives. Thepolymer blends of pGln with pLys (42000) were also among the mostpreferred adhesives. pGln and pLys (42000) by themselves exhibited goodadhesion to glass but their blends were even better adhesives.

[0113] Blends of amino acid homopolymers were most preferred asadhesives. These tests illustrate a large number of useful combinationsof amino acid polymer blends suitable for use in this present invention.The blends may be expanded to three or more polymers and includemonomers if needed to customize the adhesive characteristics to thetarget substrates.

[0114] D. Adhesiveness of Certain Polyesters:

[0115] The results of these tests with typical polyesters are summarizedin Table 4. TABLE 4 POLYESTERS Swollen pHEMA Glass max. Polymer* max.max. stress max. [Wt. Ratio] MW stress (Pa) strain (Pa) strain PLG [1:1]50,000 0 0 0 0 PLG [1:1] 1,000 28,000 3.2 23,000 2.7 PLGC-COOH 2,00020,000 2.7 28,000 4.0 [2:2:1] PLGC-COONa 2,000 30,00 4.2 27,000 3.0[2:2:1] PL 2,000 62,000 9.0 31,000 7.0

Example 2 Determination of Polymer Water Solubility

[0116] 1. Dissolve the polymer (50 mg) in tetrahydrofuran (THF) in a25-mL glass test tube.

[0117] 2. Evaporate the THF by air drying at room temperature, leaving athin film of polymer coating the bottom of the test tube.

[0118] 3. Add water (10 mL) to the test tube; mix the water and thepolymer; allow the mixture to stand at room temperature for 24 hours.

[0119] 4. Pipette the solution into a pre-weighed cup.

[0120] 5. Evaporate the water under vacuum at 40° C.

[0121] 6. Weigh the cup containing the polymer and calculate the amountof polymer in solution by subtracting the weight of the empty container.

Example 3 Matrix of PLGC With TGF-β and TCP

[0122] Reagents: TCP: DePuy, 149 μ to 250 μ diameter TGF-β₁: Genetech,0.73 mg/mL PLGC Polymer: Poly(lactide: glycolide: ε-caprolactone)(40:40:20) Na⁺ ionomer (MW 2,000) (See Preparation supra) CoatingBuffer: 20 mM Na acetate, pH 5.0 (Sigma cat # S-5889) Gelatin Buffer:2.5% Gelatin (250 mg/10 mL water), 100 Bloom General Foods Rinse Buffer:PBS pH 7.4, Boehringer Mannheim cat. 100-961 Antioxidant: 0.2% N-propylgallate in water (20 mg/10 mL; heat in microwave to place in solution)Sigma cat P-3130

[0123] Procedure:

[0124] 1. Add the desired amount of TGF-β₁ to the coating buffer (2 mL/gof TCP).

[0125] 2. Mix the TGF-β₁ coating buffer solution with dry TCP in asiliconized polypropylene container.

[0126] 3. Incubate the mixture at room temperature 3 hours withconstant, gentle mixing.

[0127] 4. Let the TCP settle or gently centrifuge; separate the TGF-β₁coating buffer by decanting.

[0128] 5. Add rinse buffer (same volume as coating buffer), mix andseparate it by decanting.

[0129] 6. Repeat rinse step.

[0130] 7. Add antioxidant solution (same volume as the rinse buffer);mix and separate it by decanting.

[0131] 8. Add gelatin buffer to the TGF-β₁-coated TCP (1.25 mL buffer/gTCP).

[0132] 9. Add TCP/buffer mixture to the viscous PLGC polymer and mix[0.796 g (44%) of polymer/1 g (56%) of TCP].

[0133] 10. Quickly freeze the matrix with liquid N₂

[0134] 11. Lyophilize the matrix.

[0135] The matrix should be stored dry at −70° C. It will readily adsorbwater from the atmosphere. The matrix can be sterilized by gammaradiation 2.5 Mrads in a N₂ atmosphere in a sealed-foil pack.

Example 4 Dissolution and Release of Matrices of Polyester Blends

[0136] This example demonstrates how the present implant matrices can bemodified to adjust degradation and delivery of a biological substance ina desired time frame.

[0137] (a) Dissolution Rate

[0138] Method: TCP (50 mg) was mixed with enough polymer to bind all theTCP. The mixture was dried completely in a vacuum oven. The dry mix wasweighed and placed in phosphate buffered saline (PBS) (5 mL). The weightof the matrix that remained bound together was measured daily.Incubation during this process was at room temp. Complete dissolutionwas defined as the point at which no matrix material remained boundtogether.

[0139] Table 5 summarizes the results of this test with varying blendsof PLG (A=12000 MW and B=500 MW). TABLE 5 DISSOLUTION STUDIES WITH PLGBLENDS A/B Ratio^(a) Days to Complete Dissolution 80/20 17 70/30 1160/40 4

[0140] Table 6 summarizes the results of tests made with random PLGCterpolymers in a ratio of 40% L-40% G-20% C with different end groups.Each terpolymer had a molecular weight of 2,000. The ionomer sampleswere prepared by carboxylating PLGC and then neutralizing thecarboxylated material with NaOH. TABLE 6 DISSOLUTION STUDIES WITH PLGCTERPOLYMERS* Polymer End Group Days to Complete Dissolution OH 50+ COO⁻50% Na⁺ 30  COO⁻ 100% Na⁺ 4

[0141] (b) Release Rate

[0142] The release of TGF-β₁ from a PLGC/TCP/TGF-β₁ matrix, prepared asdescribed in Example 3, was measured. The TGF-β₁ was extracted andassayed by ELISA as follows:

[0143] Undiluted horse serum (Sigma Cat # H-1270) and 0.02% by weightsodium azide were added to the sample. The amount of serum used dependedupon the TGF-β₁ concentration; approximately 0.4 to 1 μg TGF-β₁/mL finalconcentration was targeted. The serum and TCP were incubated for aminimum of 12 hours (overnight) at room temperature with mixing. Toremove TCP fines, the material was spun in a microfuge at 500×g for oneminute.

[0144] The samples were then assayed by an ELISA assay to determinebiological activity. The TGF-β₁ Capture ELISA protocol was as follows:Material 1. Solid support: Dynatech Immulon II, cat# 011-010-3450 2.Coating buffer: 0.05M Carbonate buffer pH 9.5 Na₂CO₂ (5.3 g/L) 3.Capture Mab: Mab <TGF-β₁> 12H5, Genentech, lot #8268-61 4. Wash buffer:PBS, 0.05% Tween 20 5. Detection Mab: Mab <TGF-β₁> 4A11-HRP Genetech,lot 16904-30 6. Standard: TGF-β₁, Genentech, used same lot as unknownsamples 7. Substrate: 3,3′,5,5′-tetramethylbenzidine (TMB), Kirkegaard &Perry Catalog #50-76-100 8. Stop solution 1M H₂SO₄

[0145] Procedure:

[0146] A 96-well microtiter plate was coated with 0.5 μg/mL of Mab 12H5in coating buffer and held at a temperature of 4° C. overnight at 100μL/well. The plate was washed with wash buffer for 6 cycles in aTitertek Microplate washer 120, and the last volume of wash buffer wasleft in the wells. The 96-well plate was incubated for 10 minutes withthe wash buffer and then emptied of the wash buffer. The TGF-β₁ sampleswere added to the washed plate and serially diluted in PBS at 100μL/well. The TGF-β₁ samples were then incubated for 1 hour at roomtemperature. The plates were again washed with wash buffer for 6 cycles.4A11-HRP conjugate was then added to the plate and diluted toapproximately 1:2000 in wash buffer, 100 μL/well. The plate was thenincubated for 1 hour at room temperature. The plates were washed withwash buffer for 6 cycles. Next, 100 μL/well of substrate was added tothe plate. The color was allowed to develop for 5 minutes. Then 50μL/well of stop solution was added. The wavelength was read at 450 nm ona Molecular Devices Vmax.

[0147] The O.D. values were curve fit using a log linear regression.Standards of diluted TGF-β₁ were used to prepare the calibration curve.The multiple needed to superimpose the regression curve on thecalibration curve at an O.D. value in the linear region was used tocalculate the unknown concentration.

[0148] The results of the tests for release of TGF-β₁ from thePLGC/TCP/TGF-β₁ matrix are summarized in Table 7: TABLE 7 RECOVERY OFTGF-β₁ FROM POLYMER MATRIX^(a) Day % Recovery of TGF-β₁ 1  42% 2 5.8% 3  1% 4  <1% 5  <1% 6  <1%

[0149] The PLGC/TCP/TGF-β₁ matrix studied in this example had a highdissolution rate (as shown in part (a), Table 6, PLGC COO⁻ Na⁺ 100%) andalso a fast release rate of TGF-β₁.

Example 5 Formulation of Ionomer/Submucosa Matrix

[0150] Reagents: TGF-β₁: Genentech 0.73 mg/mL PLGC Polymer:Poly(lactide: glycolide: ε- caprolactone) (40:40:20) Na ionomer; MW2,000 Coating Buffer: 20 mL Na acetate, pH 5.0, (Sigma); 1% gelatinfinal concentration during coating (100 Bloom General Foods)Antioxidant: 0.2% N-propyl gallate in water Small Intestinal (Preparedin accordance with Submucosa (SIS): U.S. Pat. Nos. 4,902,508 and4,956,178, supra, comminuted and lyophilized)

[0151] Procedure:

[0152] 1. Mix the desired amount of TGF-β₁ with coating buffer andsubmucosa (1 mL buffer/100 mg submucosa) to form a putty.

[0153] 2. Incubate the mixture for 1 hour at room temperature.

[0154] 3. Add antioxidant solution to the polymer and stir briefly atroom temperature until a viscous solution is produced (4 mL of 0.02% byweight of antioxidant/g of polymer).

[0155] 4. Mix the submucosa/TGF-β₁ mixture with the viscous polymersolution.

[0156] 5. Place the matrix in a container that: (a) can be frozen inliquid N₂ and (b) is shaped so the material can be coated evenly by thepolymer, i.e., a glass petri dish.

[0157] 6. Quickly freeze the matrix with liquid N₂.

[0158] 7. Lyophilize the matrix.

[0159] 8. Sterilize the polymer/matrix formulation as described inExample 3.

[0160] The polymer matrix prepared by this procedure had a finalcomposition of 67% polyester ionomer, 33% submucosa and contained 5μg/mL TGF-β₁.

Example 6 Polyester Solubility

[0161] The solubility of various polyesters was measured using theprocedure of Example 2. The results of these studies are summarized inTable 8. TABLE 8 SOLUBILITY OF POLYESTERS Polymer Ratio (MW) Solubility(g/L) PL — 200 0.01 PLG 1:1 1000 1.4 PLGC - OH 2:2:1 2000 0.2 PLGC -COOH 2:2:1 2000 0.3 PLGC - COONa 2:2:1 2000 250

Example 7 Repair of Rabbit Radius With Matrix Implant

[0162] A putty-like delivery matrix including polymer, filler and abiologically active component (TGF-β₁) (see Example 3) was evaluated invivo in the rabbit radius model.

[0163] Experimental Design:

[0164] Route of Administration:

[0165] A test article, or the autogenous control, is implanted in themidshaft radial defect.

[0166] Overview:

[0167] A 1.5-cm segment of the right radius is removed, producing aunilateral radial defect. The radial defect is implanted with a testmaterial or a control article, or receives no implant, according togroup assignment. The incision is closed, and the rabbits are allowed tosurvive for 8 weeks. At 8 weeks both radii are harvested.

[0168] Experimental Procedure:

[0169] Xylazine/ketamine cocktail is used as the anesthetic agent. Thecocktail is made by mixing xylazine (1.42 mL; 100 mg/mL) in ketamine (10mL; 100 mg/mL). The rabbits are dosed initially at approximately 0.65mL/kg I.M. (maximum of 3 mL per rabbit). An ear vein is catheterized,and additional anesthesia is given through this catheter atapproximately 0.125 of the initial dose, as needed. The right radius isclipped free of hair, then shaved or depilitated and asepticallyprepared for surgery.

[0170] Surgery:

[0171] An incision is made mid-shaft over the anterior-medial surface ofthe right forearm. Soft tissue is reflected to expose the radius. Theinterosseous ligament between the radius and the ulna is separated, andthe periosteum is excised from the radius for approximately 1.7 cm alongthe mid-shaft. A sterile spatula is placed between the radius and theulna, and a 1.5 cm segment of the radius is removed, using a saw bladeattached to a sagittal saw. The site is liberally irrigated withphysiological saline during the ostectomy to prevent overheating of thebone margins.

[0172] Experimental Sequence:

[0173] Each radial defect is filled with one of the test materials orthe autogenous graft or is left empty. After the material is molded intoposition, the soft tissue is reapposed with absorbable suture and theskin is closed with non-absorbable suture.

[0174] The amount of material actually implanted is determined byweighing the formulation after preparation, before implanting (using asterile foil weighing boat or a similar device), and then weighing thematerial not implanted.

[0175] The surgical site is radiographed to document the anatomicplacement of the material, and the rabbits are returned to their cages.Buprenorphine hydrochloride (0.15 mg SQ) is administered daily for thefirst 3 days of recovery for pain.

[0176] The rabbits are maintained post surgery for 8 weeks and thenterminated with Beuthanasia-D® Special Solution administeredintravenously. The right and left radii are removed, and soft tissue isdissected free from these bones. The operated radius is examinedhistologically for the presence of bone within the defect site(indicating a union) and the presence of cartilage, soft tissue orcracks within the defect site (indicating a possible unstable union ornon-union). The results are scored histologically according to thescale: 0=failed, 1=poor, 2=moderate, 3=good, and 4=excellent.

[0177] The results of a study made using this procedure are summarizedin Table 9. TABLE 9 RABBIT RADIUS STUDY WITH PLGC IONOMER/TGF-β₁ MATRIXAverage Treatment Score Std. Dev. n^(b) Autograft (+ control) 3.4 0.5 20Empty (− control) 0.8 1.4 20 Polymer^(a)/TCP 0 0 10Polymer^(a)/TCP/TGF-β₁ (γ-sterilized) 3.8 0.3 10

[0178] This test demonstrated that the matrices of this invention can beused to repair long bones like the radius, which contain marrow, have arich blood supply, and experience mechanical loading.

Example 8 Handing/Moldability of Polymer Matrices

[0179] The handling characteristics of the polymer implant matricesduring surgical procedures is very important. The putty-like matrixshould be moldable enough to be formed to fit into a defect site, andadhesive enough to remain in the defect site. The putty matrix shouldnot, however, be so adhesive that it will adhere easily to surfaces suchas surgeon's latex gloves or surgical instruments. This example showshow a typical polymer adhesive matrix was designed to meet theserequirements.

[0180] TCP (50 mg) was soaked with water (1 μL/mg); then polymer (SeeTable 10 for amount) was mixed with the TCP solution. The mixture wasdried with vacuum or lyophilized. The putty adhesive matrices weremeasured by the following criteria: (1) moldability—hard or soft; (2)adherence to latex gloves; and (3) adherence to instruments.

[0181] Polymers Tested:

[0182] PLG 50-50=50% lactide, 50% glycolide random copolymer

[0183] PLGC 40-50-10=40% lactide, 50% glycolide, 10% caprolactone randomterpolymer

[0184] PLGC 40-40-20=40% lactide, 40% glycolide, 20% caprolactone randomterpolymer

[0185] PLGC 40-40-20 COOH=PLGC 40-40-20 carboxylated with succinicanhydride

[0186] PLGC 40-40-20 COONa=PLGC 40-40-20 COOH neutralized with NaOH toproduce COO⁻ Na⁺ end groups

[0187] The results of the handling tests using these polymers aresummarized in Table 10. TABLE 10 HANDLING/MOLDABILITY STUDY RESULTS Amt.of Adherence polymer to gloves, Polymer Mol. Wt. (mg) Moldabilityinstruments PLG 50-50 12,000 20 No/hard No PLG 50-50 10% = 12,000 30 YesYes BLEND 90% = 400   PLG 50-50 1,400 50 No/hard No PLG 50-50 700 60 YesYes PLGC 2,000 40 No/hard No 40-50-10 PLGC 2,000 40 Yes Yes 40-40-20PLGC 2,000 40 No/hard Yes 40-40-20 COOH PLGC 2,000 35 Yes No 40-40-20COONa

[0188] This example demonstrates how changing the molecular weight,composition, and end groups influences the moldability and adherencequalities of the matrix. By lowering the molecular weight of PLG, thecomposition became more moldable, but also stickier to latex gloves.When the percentage of caprolactone was increased, the moldabilityincreased, but so did adherence to gloves. Adding the carboxyl group tothe terpolymer hardened the polymer, but neutralization of thecarboxylated terpolymer produced a moldable putty that did not adhere tolatex gloves. The PLGC 40-40-20 Na example included TCP as a filler ofsolid support. If another filler is used, the composition can betailored in a similar manner to produce a putty-like implant matrixhaving the desired characteristics.

Example 9 Effect of Glass Transition Temperature on Polymer Handling

[0189] Differential scanning calorimetry (DSC) is a commonly usedtechnique of thermal analysis. During a DSC measurement, the referencepan and sample pan are heated such that their temperature increases at aconstant predefined rate. The difference of heat flow to reference panand sample pan is measured. When the heat flow to sample pan is greaterthan that to reference pan, the measured heat flow difference isendothermic. When the heat flow to sample pan is less, the measured heatflow difference is exothermic.

[0190] DSC analysis of polymers gives information on glass transitions(T_(g)). A T_(g) is found in all amorphous polymers and in amorphousregions of partially crystalline polymers. The T_(g) of the latter isindependent of the degree of crystallization, but the magnitude of thetransition decreases with increasing crystallinity so the transitionbecomes difficult to detect in highly crystalline polymers. A polymer attemperatures above its T_(g) is limp and flexible, but a polymer belowT_(g) is brittle and stiff. (See M. C. Meikel, W. Y. Mak, S.Papaionannou, E. H. Davies, N. Mordan, J. J. Reynolds, Biomaterials,14(3), 177 (1993); and J. L. Ford, P. Timmins, “Pharmaceutical ThermalAnalysis”, Chapter 2, John Wiley & Sons, New York (1989).)

[0191] This example demonstrates that T_(g) can be a valuable tool todetermine if a polymer will remain moldable. The maximum and minimumT_(g) temperatures may be slightly different for different applicationsand or solid substrates. This example used TCP as the solid substrate.The results of these tests are summarized in Table 11. TABLE 11 GLASSTRANSITION AND HANDLING CHARACTERISTICS Polymer MW Ratio T_(g) (° C.)Handling^(a) PLG 12,000 1:1 42.9 hard Blend 12,000 1:1 20.4 hard 50% PLG4,000 1:1 50% PLG PLG 8,500 1:1 −3.1 crumbles PLG 3,500 1:1 −13.3 willmold PLGC 2,000 5:4:1 −16.0 hard but will mold PLGC 2,000 2:2:1 −28 mostmoldable Blend 500 1:1 −26.9 moldable 90% PLG 8,500 1:1 10% PLG

[0192] Modifications and variations of the polymers, implant matricesand methods of this invention will be apparent to those skilled in theart from this description. Such modifications and variations areintended to be within the scope of the appended claims.

What is claimed is:
 1. An implantable article of manufacture for use inthe release of a bioactive agent into a physiological environment, saidarticle comprising a matrix, said matrix comprising a biocompatible,bioerodable polymer, wherein the polymer has a water solubility of about0.01 to about 500 mg/mL at about 25° C. and an adhesive strength ofabout 600 to about 150,000 Pa so that the matrix is tissue adherent, anda bioactive agent.
 2. The article of claim 1 wherein the bioactive agentis a growth factor.
 3. The article of claim 1 wherein the polymer ispoly(lactide/glycolide/caprolactide).